Triggering of the antigen receptor of B or T cells leads to the initiation of multiple signalling pathways that regulate cellular proliferation and survival of immature and naive lymphocytes, and the effector functions of mature B and T cells. The signalling pathway that leads from antigen-receptor triggering to the activation of transcription factors of the nuclear factor-κB (NF-κB) family has a crucial role in these processes, and is controlled by highly similar molecular events in B and T cells. Genetic deficiencies in NF-κB-family members or signalling components that act upstream of NF-κB have been linked to immune deficiencies, whereas aberrant constitutive NF-κB activation has been associated with the development of autoimmune and neoplastic disorders. The understanding of the molecular mechanisms that control NF-κB activation in lymphocytes is therefore the focus of intense investigation. Recent studies have identified Carma1 (caspase recruitment domain, CARD, membrane-associated guanylate kinase, MAGUK, protein 1), Bcl-10 (B-cell lymphoma 10) and Malt1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) as signalling compounds that have crucial and specific roles in T-cell receptor (TCR)- and B-cell receptor (BCR)-induced NF-κB activation.
Malt1-deficient lymphocytes show impaired antigen receptor-induced activation of the transcription factor nuclear factor kappa B (NF-κB) and, as a consequence, impaired cytokine production and proliferation. Moreover, chromosomal translocation and abnormal expression or activity of Malt1 has been associated with formation of B-cell lymphomas of the mucosa-associated tissue.
More recently, the role of Bc1-10 and Malt1 has been extended to a role in lymphocyte adhesion, to activating signalling functions in other leukocytes such as phagocytes, mast cells and natural killer cells and to a signalling function downstream of G-protein coupled receptors, such as the receptors for lysophosphatidic acid or angiotensin II in non immune cells. This suggests that these proteins may have relevant immunomodulatory functions, but also be relevant for example in inflammation, tumour promotion and blood pressure regulation.
Dhaval N. Ghosalia at al., “Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray”, Biotechnology and Bioengineering, 94, 6, p. 1099-10 (2006) report enzymatic activity of 10 different plasma proteases on as many as 361 different fluorogenic substrates. The goal behind this study is to provide a microarray that gives quick information on enzymatic activity of plasma for diagnostic purposes, for example. In Table 1 a peptide substrate Ac-ARSR-ACC (ARSR: SEQ ID NO: 9) is disclosed. This reference does not concern enzymatic activity of hMalt1, the latter occurring inside cells in nature.
WO2002/33058 reports the nucleotide sequences encoding hMalt1, which is indicated as BAA83099. This reference mentions cysteine proteases, including hMalt1, and a ligand binding to the cysteine protease and inhibiting its protease activity. However, this reference does not disclose the nature of the protease activity of hMalt1 and, therefore, does not disclose a real inhibitor either.
In needs to be noted that Malt1 is known to comprise a putative active-site cysteine residue in the caspase-like domain of Malt1, as established by sequence alignments with other caspases. However, attempts to demonstrate caspase-like proteolytic activity led to the conclusion that Malt1 does not have aspartate-specific proteolytic activity (Snipas, S. J. et al., “Characteristics of the caspase-like catalytic domain of human paracaspase”, Biol. Chem. 385, 1093-1098 (2004)).
In other words, while an enzymatic activity for Malt1 was hypothesised on the basis of sequence analysis, extensive experimental efforts failed to demonstrate such a cleaving activity, which is why presently the role of hMalt1 in signalling pathways is obscure.
In view of the above, it is an objective of the present invention to elucidate the role of Carma1, Bcl-10, and/or Malt1 signalling pathways, for example in B or T cells.
It is a further objective to determine the mechanism by way of which these cell components function in the signalling pathways.
It is a still further objective to provide a possibility of interfering in this pathway so as to be able to remedy the consequences and symptoms of abnormal expression or activity of Malt1, and/or other components of this or other signalling pathways.
It is, more particularly, an objective of the present invention to provide means, such as enzyme assays or screening methods, that permit the testing or screening for bioactive principles putatively interfering in a signalling pathway of cells, such as leukocytes, and in particular lymphocytes such as B and T cells.
It is also an objective to provide research tools for assisting the search and development of bioactive principles that remedy disorders and/or diseases, such as immune deficiencies, autoimmune or neoplastic disorders. Preferably, such principles are capable of affecting the cellular proliferation and survival of lymphocytes.
It is also an objective of the present invention to provide new peptide sequences, preferably useful in research and/or medicine.
Background knowledge on the field of the present invention is found in the review publications of Thome “Carma1, Bcl-10 and Malt1 in Lymphocyte Development and Activation”, Nat. Rev. Immunol., 4, 348-359 (2004); and Rawlings et al., “The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes”, Nat. Rev. Immunol., 6, 799-812 (2006).